The ATM protein is involved in the detection and repair of genotoxic lesions. Loss of ATM function sensitizes cells to ionizing radiation, and is associated with an increased incidence of cancer. The ATM protein is therefore intimately involved in both tumor pathogenesis and tumor sensitivity to radiation therapy. The signal transduction pathway linking the detection of these genotoxic lesions to the activation of ATM is not known. We have now shown that the Tip60 Histone acetyltransferase is essential for the activation of the ATM protein in cells exposed to ionizing radiation. In response to ionizing radiation, Tip60's histone acetyltransferase activity is increased, leading to acetylation of ATM and upregulation of ATM's kinase activity. ATM and Tip60 are components of a novel DNA repair complex containing the epd scaffold protein. The long term aim is to test the hypothesis that the Tip60 Histone acetyltransferase is the key upstream regulator of the ATM protein. In Specific Aim 1, the individual protein components of the ATM complex will be identified, and the molecular interactions which govern the formation of the ATM-epc1-Tip60 complex will be determined. In Specific Aim 2, we will determine the mechanism by which Tip60 is activated by DNA damage, and test the hypothesis that the chromodomain of Tip60 interacts directly with histones at sites of DNA damage. Further, we will determine the role of Tip60 in the recruitment of ATM to sites of DNA damage. In Specific Aim 3, we will identify the lysine residue(s) on ATM which are acetylated by Tip60, determine how acetylation activates ATM, and examine how loss of ATM acetylation affects the ability of cells to detect and repair DNA lesions. This proposal will therefore characterize a newly defined signal transduction pathway in which the detection of DNA lesions is linked to the activation of downstream signaling events through acetylation. Understanding the mechanism by which genotoxic events activate ATM will provide key insights into how ATM suppresses tumor formation;and will provide information on how the inactivation of ATM in cancer predisposes individuals to cancer. Further, Tip60 and its associated proteins are potential targets for developing therapeutic agents which can modify the IR response of mammalian cells. The development of such novel therapeutics could significantly improve the clinical efficacy of radiation therapy.